[unreadable] The numerous bacterial genomes now available for select agents of significant bioterror risks should aide various studies for biodefense, if unregulated and safe genetic tools based on nonantibiotic selectable markers are available for genetic manipulations. Tools such as transposons, replicating plasmids, allelic-replacement systems, and conjugation donor strains have been critical in aiding investigators to study molecular genetics, pathogenesis, and bacteria-host interactions, which have led to vaccine, therapeutic, and diagnostic targets. For bacterial select agents, construction of multiple antibiotic resistant strains is dangerous and is restricted in the United States, which hampers studies important for biodefense and requires the need to develop non-antibiotic selectable markers. The long-term goal of this project is to develop these genetic tools, based solely on non-antibiotic marker(s), for standard repetitive manipulations of the genome of Brucella and Burkholderia species that may extend into other select agents of significant bioterrorism risks. Based on our preliminary data and ongoing research indicating that unique FRT sequences can help recycle selectable markers for almost unlimited genome manipulations, our working hypothesis and premise is that there is a requirement for only one suitable non-antibiotic selectable marker for any given species when it is coupled to the Flp-FRT excision system. Because of our success in manipulating various bacterial genomes using unique FRT sequences without undesirable deletions or rearrangements of the genome, we will extend the use of Flp-FRT to develop novel genetic tools with the mercury-resistant (MerR) for Brucella and Burkholderia species as follows: i) We will develop and test novel conjugal donor and cloning strains and shuttle-vectors for Brucella (B. abortus, B. melitensis, and B. suis) and Burkholderia (B. mallei and pseudomallei). The two non-antibiotic selectable markers that will be used are Asd (aspartate-seminaldehyde dehydrogenase) and MerR. ii) We will develop FRT-MerR-FRT cassettes that are amenable to repetitive rounds of allelic replacement and transposon mutagensis. The usefulness of these MerR-cassettes will be demonstrated in allelic-replacement experiments for B. abortus and B. mallei and transposon mutagenesis for B. pseudomallei. [unreadable] [unreadable] [unreadable]